Constructing a novel expression system by specific activation of amylase expression pathway in Penicillium

Abstract

Background: Filamentous fungi have long been used as hosts for the production of proteins, enzymes and valuable products in various biotechnological applications. However, recombinant proteins are expressed with highly secreted host proteins when stronger promoters are used under inducing conditions. In addition, the efficiency of target protein expression can be limited by the application of constitutive promoters in recently developed filamentous fungal expression systems. Results: In this study, a novel expression system was constructed by using a Penicillium oxalium strain that has powerful protein secretion capability. The secretory background of the host was reduced by knocking out the Amy13A protein and utilizing the starch as a carbon source. The strong promoter amy15A(p) was further improved by overexpressing the transcription activator AmyR and deleting of putative repressor CreA. By using the native amylase Amy15A as a reporter, the efficiency of expression from the amy15A promoter was dramatically and specifically enhanced after redesigning the regulatory network of amylase expression. Conclusions: Our researches clearly indicated that the triple-gene recombinant strain Δ13A-OamyR-ΔCreA, with the amy15A(p) promoter could be used as a suitable expression system especially for high-level and high-purity protein production.

An impact of N-glycosylation on biochemical properties of a recombinant α-amylase from Bacillus licheniformis

“…In practice, glycosylation is a convenient tool for protein engineering applications, because in this way, it is possible to modify biochemical characteristics of the enzyme. Protein glycosylation has been observed during protein expression in yeasts Saccharomyces cerevisiae , P.pastoris, and Kluyveromyces lactis [ 15 , [33] , [34] , [35] , [36] ] and in filamentous fungi of genera Aspergillus, Penicillium, and Trichoderma [ 37 , 38 ]. Although S. cerevisiae is characterized by abundant glycosylation, the proteins expressed in P. pastoris have moderate glycosylation [ 39 ].…”

Anticancer Asparaginases: Perspectives in Using Filamentous Fungi as Cell Factories

“…The classic first approach to increase the level of recombinant proteins to be secreted is to increase the recombinant gene expression levels. This can be achieved by the use of strong promoters, e.g., amylase promoters [39]. However, high gene-expression levels can result in high demands in the ER of protein folding [40], often disrupting ER homeostasis an resulting in stress in the organelle where misfolded proteins are accumulated, triggering a feedback mechanism termed unfolded protein response (UPR) [41].…”

Avaliação do potencial antimicrobiano dos extratos de fungos Penicillium spp.

“…Este resultado revela o elevado potencial dos compostos químicos produzidos por esta espécie para estudos envolvendo a prospecção da atividade antimicrobiana. Ao consultar a literatura, constatou-se que as pesquisas envolvendo o P. oxalicum estão concentradas na obtenção de pigmentos (YAO, et al, 2016), prospecção enzimática de amilases (Pi, et al, 2020), celulases (Bomtempo, et al, 2017), hemicellulases (Song, et al, 2016) quitinases (Rodríguez et al, 1995) e pectinases (Kumar & Suneetha, 2021).…”